Protein Cys Research Articles

Molecular Basis of the Mechanism of Thiol Oxidation by Hydrogen Peroxide in Aqueous Solution: Challenging the SN2 Paradigm

The oxidation of cellular thiol-containing compounds, such as glutathione and protein Cys residues, is considered to play an important role in many biological processes. Among possible oxidants, hydrogen peroxide (H(2)O(2)) is known to be produced in many cell types as a response to a variety of extracellular stimuli and could work as an intracellular messenger. This reaction has been reported to proceed through a S(N)2 mechanism, but despite its importance, the reaction is not completely understood at the atomic level. In this work, we elucidate the reaction mechanism of thiol oxidation by H(2)O(2) for a model methanethiolate system using state of the art hybrid quantum-classical (QM-MM) molecular dynamics simulations. Our results show that the solvent plays a key role in positioning the reactants, that there is a significant charge redistribution in the first stages of the reaction, and that there is a hydrogen transfer process between H(2)O(2) oxygen atoms that occurs after reaching the transition state. These observations challenge the S(N)2 mechanism hypothesis for this reaction. Specifically, our results indicate that the reaction is driven by a tendency of the slightly charged peroxidatic oxygen to become even more negative in the product via an electrophilic attack on the negative sulfur atom. This is inconsistent with the S(N)2 mechanism, which predicts a protonated sulfenic acid and hydroxyl anion as stable intermediates. These intermediates are not found. Instead, the reaction proceeds directly to unprotonated sulfenic acid and water.

Tyrosinenitration affects thymidylate synthase properties

Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

Redox biology: computational approaches to the investigation of functional cysteine residues.

Cysteine (Cys) residues serve many functions, such as catalysis, stabilization of protein structure through disulfides, metal binding, and regulation of protein function. Cys residues are also subject to numerous post-translational modifications. In recent years, various computational tools aiming at classifying and predicting different functional categories of Cys have been developed, particularly for structural and catalytic Cys. On the other hand, given complexity of the subject, bioinformatics approaches have been less successful for the investigation of regulatory Cys sites. In this review, we introduce different functional categories of Cys residues. For each category, an overview of state-of-the-art bioinformatics methods and tools is provided, along with examples of successful applications and potential limitations associated with each approach. Finally, we discuss Cys-based redox switches, which modify the view of distinct functional categories of Cys in proteins.

Conformational Changes during Kinase On-Off Switching in the Chemosensory Signaling Array of the Bacterial Cell Membrane: Detection by One-Sample FRET

Fluorescence resonance energy transfer (FRET) is a powerful tool to study macromolecular assemblies in vitro under near physiological conditions. One Sample FRET (OS-FRET) employs a novel, non-fluorescent methanethiosulfonate-linked acceptor that can be reversibly coupled to a target protein Cys residue (Erbse et al, submitted). The design of OS-FRET provides distinct advantages over existing methods for quantitative FRET measurements in virtually any fluorimeter or detection device. We previously demonstrated the utility of the method by applying it to a soluble complex formed by the CheA and CheW proteins of the bacterial chemosensory pathway.

Cysteine Function Governs Its Conservation and Degeneration and Restricts Its Utilization on Protein Surfaces

Cysteine (Cys) is an enigmatic amino acid residue. Although one of the least abundant, it often occurs in the functional sites of proteins. Whereas free Cys is a polar amino acid, Cys in proteins is often buried, and its classification on the hydrophobicity scale is ambiguous. We hypothesized that the deviation of Cys residues from the properties of a free amino acid is due to their reactivity and addressed this possibility by examining Cys in large protein structure data sets. Compared to other amino acids, Cys was characterized by the most extreme conservation pattern, with the majority of Cys being either highly conserved or poorly conserved. In addition, clustering of Cys with another Cys residue was associated with high conservation, whereas exposure of Cys on protein surfaces was associated with low conservation. Moreover, although clustered Cys behaved as polar residues, isolated Cys was the most buried residue of all, in disagreement with known chemical properties of Cys. Thus, the anomalous hydrophobic behavior and conservation pattern of Cys can be explained by elimination of isolated Cys from protein surfaces during evolution and by clustering of other Cys residues. These findings indicate that Cys abundance is governed by Cys function in proteins rather than by the sheer chemical–physical properties of free amino acids, and suggest that a high tendency of Cys to be functionally active can considerably limit its abundance on protein surfaces.

Characterization of Surface-Exposed Reactive Cysteine Residues in Saccharomyces cerevisiae

Numerous cellular processes are subject to redox regulation, and thiol-dependent redox control, acting through reactive cysteine (Cys) residues, is among the major mechanisms of redox regulation. However, information on the sets of proteins that provide thiol-based redox regulation or are affected by it is limited. Here, we describe proteomic approaches to characterize proteins that contain reactive thiols and methods to identify redox Cys in these proteins. Using Saccharomyces cerevisiae as a eukaryotic model organism, we identified 284 proteins with exposed reactive Cys and determined the identities of 185 of these residues. We then characterized subsets of these proteins as in vitro targets of major cellular thiol oxidoreductases, thioredoxin and glutaredoxin, and found that these enzymes can control the redox state of a significant number of thiols in target proteins. We further examined common features of exposed reactive Cys and compared them with an unbiased control set of Cys using computational approaches. This analysis (i) validated the efficacy of targeting exposed Cys in proteins in their native, folded state, (ii) quantified the proportion of targets that can be redox regulated via thiol oxidoreductase systems, and (iii) revealed the theoretical range of the experimental approach with regard to protein abundance and physicochemical properties of reactive Cys. From these analyses, we estimate that approximately one-fourth of exposed Cys in the yeast proteome can be regarded as functional sites, either subject to regulation by thiol oxidoreductases or involved in structural disulfides and metal binding.

Abstract 1324: Modulation of IR induced NFKB activity by manipulation of NOS coupling

Abstract The transcriptional activity of NFκβ is in part regulated by nitric oxide (NO) and reactive nitrogen species (RNS). Previously we demonstrated that low doses of ionizing radiation (IR) activate constitutive, Ca2+ dependent NO Synthases (NOS) and the generation of RNS, resulting in protein Tyr nitration and/or Cys s-nitrosylation. Previous studies in our lab demonstrated that nitration of IκBα is critical for the IR-induced activation of NFκβ whereas others have shown that s-nitrosylation of the p65 subunit leads to the inhibition of NFκβ. Here we show that exposing MCF-7 cells to 5Gy IR results not only in Tyr nitration of IκBα but also Cys s-nitrosylation of NFκβ both with a maximum at 30min. post IR. One explanation for the discordant results is that NOS activity is uncoupled, resulting in the production of increased levels of superoxide anion as opposed to NO. Tetrahydrobiopterin (BH4) is a necessary cofactor that stabilizes the enzyme and allows the generation of NO through oxidation of L-arginine. However, if BH4 levels become limiting as found in chronic inflammatory or oxidative conditions, electron transfer is instead shuttled to molecular oxygen resulting in the production of superoxide anion, a scenario in which NOS is described as “uncoupled.” When NOS is uncoupled the resulting increase in superoxide can lead to enhanced levels of peroxynitrite formation, resulting in enhanced IκBα Tyr nitration and NFκβ activity. This evidence has led to the hypothesis that by altering cellular levels of BH4, the levels of Tyr nitration and Cys s-nitrosylation can be manipulated, leading to changes in NFκβ activity. To determine the effects of exogenous BH4 on IκBα nitration in MCF-7 cells, cells were treated with varying concentrations of cell permeable sepiapterin (SP), a precursor of BH4, and exposed to 5Gy IR. The results show that between 100nM-500nM IκBα nitration is all but completely eliminated, resulting in decreased NFκβ activity as assessed by luciferase assay. Initial experiments looking at the effects of SP on p65 s-nitrosylation have been variable. At a minimum, levels of s-nitrosylation remain the same as untreated cells; however, enhancement of s-nitrosylation is seen in some experiments. Studies are ongoing to clarify SP's effects on p65 s-nitrosylation as well as studies to determine SP's effects on tumor cell survival post IR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1324.

Thioredoxin interacting protein (Txnip) is feedback regulator of S‐nitrosylation

Nitric oxide exerts a plethora of biological effects via protein S‐nitrosylation, a redox‐based reaction that converts a protein Cys thiol to a S‐nitrosothiol. However, while the regulation of protein S‐nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus‐coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin‐interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin‐mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S‐nitrosylation.

Thioredoxin-interacting Protein (Txnip) Is a Feedback Regulator of S-Nitrosylation

Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation. We now demonstrate that protein denitrosylation by thioredoxin is regulated dynamically by thioredoxin-interacting protein (Txnip), a thioredoxin inhibitor. Endogenously synthesized nitric oxide represses Txnip, thereby facilitating thioredoxin-mediated denitrosylation. Autoregulation of denitrosylation thus allows cells to survive nitrosative stress. Our findings reveal that denitrosylation of proteins is dynamically regulated, establish a physiological role for thioredoxin in protection from nitrosative stress, and suggest new approaches to manipulate cellular S-nitrosylation.

Assays for S -Nitrosothiols and S -Nitrosylated Proteins and Mechanistic Insights Into Cardioprotection

Gender differences in the incidence of cardiovascular disease may be ascribed at least in part to the protective effects of estrogen through both long-term and rapid (“nongenomic”) actions.1 Nitric oxide (NO), generated by endogenous cardiac NO synthases (NOS; NOS1 or neuronal NOS [nNOS], NOS2 or inducible NOS, NOS3 or endothelial NOS), plays a major role in both normal cardiac physiology and cardioprotection (particularly myocardial ischemia/reperfusion injury; see the Figure),2 and the article by Lin et al3 in this issue of Circulation contributes to growing evidence that the cardioprotective functions of estrogen are conveyed in significant part by NO. A rapidly expanding body of studies indicates that NO acts in most cellular contexts largely through the covalent modification of protein Cys thiols (to generate an S -nitroso [SNO]-protein, designated S -nitrosylation),4 and recent analyses using a new generation of analytical approaches (see the Table) both confirm original measurements of SNO-proteins that have been long-standing sources of controversy in the field and point to important roles for S -nitrosylation in NO-derived cardioprotection (see the Figure).3,5,6 Figure. NO-based mechanisms for preconditioning in ischemia/reperfusion injury. Accumulating evidence suggests a role for NO/SNO in estrogen and adrenergic receptor–mediated and statin-induced preconditioning in ischemia/reperfusion injury. Protein S -nitrosylation, resulting from increased NOS expression and activity and altered subcellular localization, appears to be a principal mediator of these effects. View this table: Table. Methodologies for the Detection and Absolute or Relative Quantification of SNO-Proteins and Other S -Nitrosothiols Article see p 245 In the myocardium, endothelial NOS is associated primarily with sarcolemmal caveolae and perhaps β-arrestin, and endothelial NOS–derived NO influences β-adrenergic receptor stimulation of myocardial contractility,7 at least in part through S -nitrosylation and inhibition of the L-type Ca2+ channel5 and G-protein receptor kinase 2.8 In contrast, nNOS …

A Genetic Analysis of Nitrosative Stress

Nitrosative stress is induced by pathophysiological levels of nitric oxide (NO) and S-nitrosothiols (e.g., S-nitrosoglutathione, GSNO) and arises, at least in significant part, from the nitrosylation of critical protein Cys thiols (S-nitrosylation) and metallocofactors. However, the mechanisms by which NO and GSNO mediate nitrosative stress are not well understood. Using yeast Saccharomyces cerevisiae strains lacking NO- and/or GSNO-consuming enzymes (flavohemoglobin and GSNO reductase, respectively), we measured the individual and combined effects of NO and GSNO on both cell growth and the formation of protein-bound NO species. Our results suggest an intracellular equilibrium between NO and GSNO, dependent in part on cell-catalyzed release of NO from GSNO (i.e., "SNO-lyase" activity). However, whereas NO induces multiple types of protein-based modifications, levels of which correlate with inhibition of cell growth, GSNO mainly affects protein S-nitrosylation, and the relationship between S-nitrosylation and nitrosative stress is more complex. These data support the idea of multiple classes of protein-SNO, likely reflected in divergent routes of synthesis and degradation. Indeed, a significant fraction of protein S-nitrosylation by NO occurs in the absence of O(2), which is commonly assumed to drive this reaction but instead is apparently dependent in substantial part upon protein-bound transition metals. Additionally, our findings suggest that nitrosative stress is mediated principally via the S-nitrosylation of a subset of protein targets, which include protein SNOs that are stable to cellular glutathione (and thus are not metabolized by GSNO reductase). Collectively, these results provide new evidence for the mechanisms through which NO and GSNO mediate nitrosative stress as well as the cellular pathways of protein S-nitrosylation and denitrosylation involving metalloproteins, SNO lyase(s) and GSNO reductase.

Reduced reliance on the trace element selenium during evolution of mammals.

Evolution from fish to mammals was accompanied by decreased use of selenocysteine, raising questions about the need for selenium dietary supplements when pathology is not imminent.

Unbiased identification of cysteine S-nitrosylation sites on proteins

Covalent addition of nitric oxide (NO) to Cys-sulfur in proteins, or S-nitrosylation, plays pervasive roles in the physiological and pathophysiological modulation of mammalian protein functions. Knowledge of the specific protein Cys residues that undergo NO addition in different biological settings is fundamental to understanding NO-mediated signal transduction. Here, we describe in detail an MS-based proteomic protocol for facile, high-throughput and unbiased discovery of SNO-Cys residues in proteins from complex biological samples. The approach, termed SNOSID (SNO-Cys site identification), can be used to identify endogenous and chemically induced S-nitrosylation sites in proteins from tissues or cells. Identified SNO-Cys sites may provide insights into novel mechanisms and proteins that mediate NO bioactivities in health and disease. SNOSID builds on the biotin-switch method for covalent addition of disulfide-linked biotin at S-nitrosylation sites on proteins. Biotinylated proteins are then subjected to trypsinolysis and the resulting biotin-tagged peptides are affinity-captured on streptavidin-agarose. After selective elution with beta-mercaptoethanol, the peptides are sequenced using nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). Validation that identified peptide ions as originating from authentic NO-Cys-containing precursor proteins can be provided by establishing that these peptide ions are absent from control samples where S-NO bonds were subjected to prior photolysis, using a UV transilluminator. The protocol requires approximately 2 days for sample processing, including the incubation time for proteolysis. An additional 1-2 days is needed for sample analysis by nLC-MS/MS and data analysis/interpretation.

Balancing reactivity against selectivity: The evolution of protein S-nitrosylation as an effector of cell signaling by nitric oxide

Produced by the action of lightning in the atmosphere of the pre-biotic earth, nitric oxide (NO) is a free radical molecule that provided the major nitrogen source for development of life. Remarkably, when atmospheric sources of NO became restrictive, organisms evolved the capacity for NO biosynthesis and NO took on bioregulatory roles. We now recognize NO as an ancestral regulator of diverse and important biological functions, acting throughout the phylogenetic tree. In mammals, NO has been implicated as a pivotal regulator of virtually every major physiological system. The bioactivities of NO, and reactive species derived from NO, arise predominantly from their covalent addition to proteins. Importantly, S-nitrosylation of protein cysteine (Cys) residues has emerged as a preeminent effector of NO bioactivity. How and why NO selectively adds to particular Cys residues in proteins is poorly understood, yet fundamental to how NO communicates its bioactivities. Also, evolutionary pressures that have shaped S-nitrosylation as a biosignaling modality are obscure. Considering recently recognized NO signaling paradigms, we speculate on the origin of NO signaling in biological systems and the molecular adaptations that have endowed NO with the ability to selectively target a subset of protein Cys residues that mediate biosignaling.

Assessment and Application of the Biotin Switch Technique for Examining Protein S-Nitrosylation under Conditions of Pharmacologically Induced Oxidative Stress

Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.

High-Throughput Identification of Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redox-active Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox-active Cys in these proteins.

A Dual Bcr-Abl/Lyn Tyrosine Kinase Inhibitor, NS-187, Is a Novel Agent for Imatinib-Resistant Leukemia.

Chemical modifications of imatinib mesylate made with the guidance of molecular modeling yielded several promising compounds. Among them, we selected a compound denoted NS-187 (elsewhere described as CNS-9) on the basis of its affinity to Abl, and also to Lyn, which may be involved in imatinib-resistance (Figure). The most striking structural characteristic of NS-187 is its trifluoromethyl (CF3) group at position 3 of the benzamide ring. The presence of the CF3 group strengthened the hydrophobic interactionss of the molecule with the hydrophobic pocket of Abl. Another possible merit of the CF3 group is that it may fix the conformation of the drug by hindering its rotation at the 4-position of the benzamide ring; as a result, a CF3-bearing molecule may be more potent than more flexible compounds such as imatinib. In fact, NS-187 was 25–55 times more potent than imatinib in vitro and and at least 10 times more potent than in vivo. NS-187 also inhibited the phosphorylation and growth of all Bcr-Abl mutants tested except T315I at physiological concentrations. Another special feature of NS-187, in addition to its increased affinity to Abl is its unique spectrum of inhibitory activity against protein kinases. At a concentration of 0.1 μM, NS-187 inhibited only four of 79 tyrosine kinases, that is, Abl, Arg, Fyn, and Lyn. Notably, at 0.1 μM NS-187 did not inhibit PDGFR, Blk, Src or Yes. The IC50 values of NS-187 for Abl, Src and Lyn were 5.8 nM, 1700 nM and 19 nM, respectively, and those of imatinib were 106 nM, >10,000 nM and 352 nM, respectively. These findings indicate that NS-187 acts as a Bcr-Abl/Lyn inhibitor. In this respect, NS-187 may stand out among other novel Abl tyrosine kinase inhibitors, because BMS-354825 inhibits all members of the Src family, while AMN-107 inhibits none of the Src-family kinases. Our proposed docking models of the NS-187/Abl complex support the notion that NS-187 is more specific for Lyn than for Src. The amino acid at position 252 is either Gln or Cys in Src-family proteins. NS-187 inhibited the Gln252-bearing proteins Abl, Fyn and Lyn but had lower activity against the Cys252-bearing Src and Yes. This is probably because Gln, unlike Cys, readily forms hydrogen bonds. The distinguishing characteristic of NS-187, its high affinity for and specific inhibition of Abl and Lyn, may be useful in the treatment of Bcr-Abl-positive leukemia patients. [Display omitted]

Zn Protein Simulations Including Charge Transfer and Local Polarization Effects

Nearly half of all proteins contain metal ions, which perform a wide variety of specific functions associated with life processes. However, insights into the local/global, structural and dynamical fluctuations in metalloproteins from molecular dynamics simulations have been hampered by the "conventional" potential energy function (PEF) used in nonmetalloprotein simulations, which does not take into the nonnegligible charge transfer and polarization effects in many metal complexes. Here, we have carried out molecular dynamics simulations of Zn(2+) bound to Cys(-) and/or His(0) in proteins using both the conventional PEF and a novel PEF that accounts for the significant charge transfer and polarization effects in these Zn complexes. Simulations with the conventional PEF yield a nontetrahedral Cys(2)His(2) Zn-binding site and significantly overestimate the experimental Zn-S(Cys(-)) distance. In contrast, simulations with the new PEF accurately reproduce the experimentally observed tetrahedral structures of Cys(2)His(2) and Cys(4) Zn-binding sites in proteins, even when the simulation started from a nontetrahedral Zn(2+) configuration. This suggests that simulations with the new PEF could account for coordinational changes at Zn, which occurs during the folding/unfolding of Zn-finger proteins and certain enzymatic reactions The strategy introduced here can easily be applied to investigate Zn(2+) interacting with protein ligands other than Cys(-) and His(0). It can also be extended to study the interaction of other metals that have significant charge transfer and polarization effects.

A trojan horse approach for silencing thymidylate synthase.

In this paper we present a new and possibly more effective way of inhibiting thymidylate synthase (TS) in cells than through the use of substrate analogue inhibitors. An inactive double mutant of TS (DM), Arg(126)Glu/Cys(146)Trp, is shown to progressively impair the reactivation of native Escherichia coli TS when the two are denatured together in vitro. The individual single mutant proteins Arg(126)Glu and Cys(146)Trp showed little or no inhibition. When the DM is introduced into E. coli and induced from an expression plasmid, the mutant subunits act as a decoy in deceiving newly formed native TS subunits to fold with them to yield inactive heterodimers. As a consequence of the depletion of TS, the cells die a "thymineless" death when grown in medium devoid of thymine. Addition of thymine to the medium enables the cells to grow normally, although only very low levels of TS activity could be detected in those cells containing induced DM. The individual single-site mutations of the DM, Arg(126)Glu and Cys(146)Trp, did not inhibit growth, as might be expected from the in vitro studies. However, when a nontoxic level of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) is added to growing DM-transformed cells, the combination is lethal to the cells. These experiments suggest that a similar dominant-negative response to the DM of TS could be affected in tumor cells, for which preliminary evidence is presented. This technique, either alone or combined with other modalities, suggest a new approach to targeting cells for chemotherapy.

DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.